Identification and characterization of rare invariant CD1d-restricted NKT cells by nine-color flow cytometry. Top panels show the gating to identify CD3+ single lymphocytes, using SSC-Area versus SSC-Height (upper left panel), followed by gating on lymphocytes (upper middle panel), and CD3+ cells (upper right panel). NKT cells were identified among CD3+ cells as staining double positive for αGalCer-loaded CD1d DimerX and anti-Vα24, and further subdivided as CD4-CD161+, CD4+CD161+, and CD4+CD161-. In subset analysis the zebra plot color follows the gate color upstream in the gating scheme. CD1d DimerX was detected with anti-mouse IgG1 PE, biotinylated anti-CD127 was detected with streptavidin-conjugated QD605. Other reagents include: Anti-CD57 FITC, anti-CD161 PE-Cy5, anti-CD56 PE-Cy7, anti-CD16 PacB, anti-CD3 CasY, anti-Vα24 APC, anti-CD4 APC-Cy7.